Control of intracellular pH transmembrane proton gradients is essential for all forms of life. Normal and pathophysiological processes may lead to cellular acidification, which both directly and indirectly modulates the function of a wide variety of cellular proteins and signaling pathways. A central function of voltage-gated proton channels is to provide an efflux pathway for excess H+. In 2006, we and others identified the Hvcn1 gene and described the function of the encoded Hv1 voltage-gated proton channel. Hv1 is sufficient to reconstitute the hallmark biophysical properties of the native voltage-gated H+ conductance when expressed in heterologous systems. Hv1 is also required for expression of voltage-gated H+ currents in leukocytes. The identification of Hv1 facilitates the investigation of fundamentally important questions of proton channel structure and mechanism of action. Like homologous voltage sensor domain (VSD)-containing proteins such as voltage-gated Ca2+, K+ and Na+ channels and voltage-sensitive phosphatases, Hv1 is activated by membrane depolarization. However, in contrast to other VSD proteins, the opening of Hv1 channels is also controlled by the transmembrane pH gradient (i.e., net intracellular acidification shifts voltage-dependent activation toward negative potentials). When open, Hv1 selectively allows protons to flow down their electrochemical gradient to dissipate any existing outwardly-directed pH gradient and thereby promote net intracellular alkalinization. A molecular understanding of the mechanisms that are responsible for the control of Hv1 channel opening in response to voltage and pH gradients is lacking. Likewise, the structural determinants in the Hv1 VSD that are required to form the aqueous 'water-wire' H+ permeation pathway in Hv1 are not known. We will utilize a combination of site-directed mutagenesis, voltage clamp electrophysiology and fluorimetry, and computer- aided modeling and simulation of the Hv1 protein structure to answer fundamental questions about molecular mechanisms in the Hv1 proton channel. A more detailed understanding of Hv1 functional properties will pave the way for the development of novel pharmacological and genetic therapies designed to treat diseases that are caused by aberrant control of Hv1 proton channel activity.